The present description refers to a method for immuno-histochemical staining of a formalin-fixed, paraffin-embedded tissue section comprising the steps of a) providing a solid support, b) mounting the formalin-fixed, paraffin-embedded tissue section onto the solid support, c) removing the paraffin from the formalin-fixed, paraffin-embedded tissue section, d) heating the tissue section mounted on the solid support to retrieve epitopes at 50 to 70° C. for 12 to 24 h, and e) staining the tissue section mounted on the solid support, wherein at least step e) is performed in the presence of 0.5 to 3.0 M sodium chloride. The present description further refers to a kit for performing the method.
Formalin fixation and paraffin embedding (FFPE) is a well-known procedure for tissue fixation for diagnostic histology and long term storage in clinical routine. Large archives of FFPE tissue (FFPET) samples are used for biomarker discovery studies as well as early clinical studies. Furthermore, FFPET samples can be used for isolation of RNA which can further be applied in gene expression analyses. Although, RNA from FFPET samples shows a high degree of degradation, it is still sufficient for RT-qPCR analyses provided that a sufficient amount of material is used for RNA isolation. Such analyses can be used to generate a first biomarker hypothesis and simply for hypothesis testing (Lohmann et al, Methods. 2013 January; 59(1)).
To be able to separate blood vessels and tumor cell nests from FFPE tissue sections and perform RT-qPCR analysis, we combined the following technologies: laser capture micro-dissection (LCM) from FFPET after immuno-histochemical staining with RT-qPCR analysis after pre-amplification of the cDNA.
The challenges the skilled person is faced with when combining LCM from IHC-stained FFPET sections followed by RT-qPCR analysis is the very limited amount of material after LCM, RNA degradation after fixation of the sample tissue and in addition the RNA degradation during the immuno-histological staining procedure. The classical staining procedure includes epitope retrieval (e.g. HIER protocol at 98° C.) and incubation times with buffers that cannot be produced under RNAse free conditions (antibody solution, washing buffers, dyes). The described RNA degradation in combination with the very limited amount of material after LCM is the major problem of this workflow if the RNA is intended to be used in RT-qPCR analyses. The degradation of RNA lead to unreliable results in gene expression analyses following LCM, e.g. the ratio between a biomarker of interest and a reference gene. mRNA stability is gene-specific and therefore they are degrading to a different extend, leading to wrong results in relative quantification for gene expression analysis.
To overcome this problem of RNA degradation, different procedures have been established to keep the degradation to a minimum. The majority of publications for example use Fresh Frozen (FF) tissue which has the advantage of less degradation processes when used for RT-qPCR analyses. The major drawback, however, is the fact that FF tissue is a largely limited source as compared to FFPET (Buckanovich et al., Cancer Biol Ther. 2006 June; 5(6):635-42; Gjerdrum et al., Diagn Mol Pathol 2004 (13), p. 224-233) It is often difficult to obtain a sufficient amount of FF tissue for performing a study leading to statistically significant results. Furthermore the clinical sample material available for retrospective testing is typically FFPET.
Therefore, a main focus in this field was the development of techniques using FFPET in combination with LCM and RT-qPCR analyses minimizing the RNA degradation. Ultra-fast staining procedures for FFPET-sections were developed to minimize RNA degradation during the staining. However, analyzing the RNA from such micro-dissected, ultra-fast stained FFPET-sections showed that after antibody incubation with very short incubation periods (2×5 min) still approximately 90% of the RNA was degraded as compared to a simple histological stain (Hematoxylin) (Gjerdrum et al., Diagn Mol Pathol 2004 (13)). In addition, ultra-fast staining protocols are not applicable to all antibodies as some need several hours to overnight incubation (Brown et al., RNA Journal 2009 (15), p. 2364-2374). Such long term incubation procedures lead to massive RNA degradation, such that gene expression analyses are simply impossible.
Therefore, the object of the present description is to provide an improved staining technique for FFPET-sections subsequently used for RNA isolation, wherein the improved staining technique leads to a yield and quality of the isolated RNA from FFPET-sections sufficient for RNA analyses, such as gene expression analyses.